Despite nearly 50 human subjects enrolled in seven hemophilia gene therapy trials over 10 years,1 gene therapy is still not a feasible alternative for patients with hemophilia. In situ correction has now been successfully accomplished in an animal — specifically, in the hemophilia B mouse. Katherine High and her colleagues from Children’s Hospital of Philadelphia, for the first time, have successfully inserted a normal F9 gene into the genome of a hemophilia B mouse using zinc finger nucleases (ZFNs) to accomplish genome editing and correction of the mutated gene in situ. ZFNs are fusion proteins that recognize specific DNA sequences in a gene to induce site-specific double-strand DNA breaks to allow insertion of a normal gene at that locus.2

To accomplish the in vivo genome editing in the hemophilia B mouse model the investigators designed a ZFN targeting intron 1 of the human F9 gene. This approach was based on the fact that nearly all of the mutations causing hemophilia B are distributed across the coding sequences of exons 2-8. Thus, by targeting intron 1, the investigators hypothesized that they would be able to correct nearly all, if not all, of the disease-causing mutations in the human F9 gene.

In their in vitro studies, High’s group demonstrated that ZFN efficiently induced double-strand breaks at a locus in intron 1, and that when co-delivered with a gene-targeting vector this ZFN could insert the normal F9 gene at the specific locus targeted in exon 1. Because ZFN targets only the human F9 gene, the investigators engineered a mouse to carry a mutated human F9 gene. To correct the mutated human F9 gene, the ZFN was coadministered by intraperitoneal injection with an AAV-8 donor vector and a cDNA cassette containing exons 2-8 of the wild-type human F9 gene.

Human factor IX activity levels detected in mouse plasma two days later by ELISA assay averaged 3 to 7 percent of normal. The degree of correction of factor IX levels was noted to be dependent on the dose of AAV-donor: the higher the vector dose, the higher the level. Several days after ZFN injection in the mouse, hepatectomy was performed to demonstrate stable persistence of factor IX levels after the “gene editing.” There was an accompanying correction of the hemophilia B phenotype, demonstrated by a shortening of the APTT, from mid-60s to the normal range, mid-30s.

So, is this approach ready for use in humans? Unlike vector-based gene therapies currently under clinical investigation, ZFN-directed gene editing would produce a permanent correction after a single treatment (presumably intravenous in humans rather than the intraperitoneal route used in mice); and if proven safe and effective in dogs and in phase I/II trials in humans, it might be a potential approach. Is ZFN-directed genome editing of a mutated F9 gene sufficient to result in normal hemostasis? In the mouse model, this approach achieved a factor IX level high enough to prevent spontaneous bleeding. If it resulted in similar levels in man, this would also prevent spontaneous bleeding, which accounts for the major morbidity of the disease. Will this approach be safe? Dog studies, followed by careful phase I clinical trials in humans, would be required to answer this question. For humans, the critical steps will be to analyze any potential integration sites in the human genome in addition to intron 1 of the F9 gene. Will there be an immune response to AAV? The answer to this question will require prospective assessment with careful monitoring of liver function tests to detect the transient transaminitis that may accompany exposure to some AAV vectors. It is of note that small studies indicate that short-term immunosuppression with mycophenolate and rapamycin may alleviate or prevent this problem.3 Will it be possible to escalate AAV vector dosing to achieve higher factor IX levels? This will require careful escalation of vector dose in future clinical trials.